The present invention relates to the field of molecular biology and the genetic transformation of plants with foreign gene fragments. More particularly, the invention relates to a binary expression system useful for conditionally expressing transgenes in plants.
Two serious technical problems beset plant transgenics. First, plant transgene expression attains only low and inconsistent levels. These poor expression levels are attributable in part to random chromosomal integration (xe2x80x98position effectsxe2x80x99) and in part to a general lack of gene copy number-dependent expression. Episomal vectors are expected to overcome these problems. In constrast to plants, microbes can attain high-level expression through episomal (plasmid) vectors because these vectors can be maintained by selection. Although plant viruses have been used as episomal expression vectors, their use has been restricted to transient expression because of lack of selection and/or their cellular toxicity (U.S. Pat. No. 4,855,237, WO 9534668).
Second, non-specific expression of transgenes in non-desired cells and tissues hinders plant transgenic work. This is important where the goal is to produce high levels of phytotoxic materials in transgenic plants. Conditional transgene expression will enable economic production of desired chemicals, monomers, and polymers at levels likely to be phytotoxic to growing plants by restricting their production to production tissue of transgenic plants either just prior to or after harvest. Therefore, lack of a commercially usable conditional expression system and the difficulty in attaining a reliable, high-level expression both limit development of transgene expression in plants.
Plant Viruses
Viruses are infectious agents with relatively simple organization and unique modes of replication. A given plant virus may contain either RNA or DNA, and may be either single- or double-stranded.
RNA Plant Viruses
Double-stranded RNA plant viruses include rice dwarf virus (RDV) and wound tumor virus (WTV). Single-stranded RNA plant viruses include tobacco mosaic virus (TMV) and potato virus X (PVX), turnip yellow mosaic virus (TYMV), rice necrosis virus (RNV) and brome mosaic virus (BMV). The RNA in single-stranded RNA viruses may be either a plus (+) or a minus (xe2x88x92) strand.
Although many plant viruses have RNA genomes, organization of genetic information differs between groups (the major groupings designated as monopartite, bipartite and tripartite). The genome of most monopartite plant RNA viruses is a single-stranded molecule of (+)-sense. There are at least 11 major groups of viruses with this type of genome. Examples of this type of virus are TMV and PVX. At least six major groups of plant RNA viruses have a bipartite genome. In these, the genome usually consists of two distinct (+)-sense single-stranded RNA molecules encapsidated in separate particles. Both RNAs are required for infectivity. Cowpea mosaic virus (CPMW) is one example of a bipartite plant virus. A third major group, containing at least six major types of plant viruses, is tripartite, with three (+)-sense single-stranded RNA molecules. Each strand is separately encapsidated, and all three are required for infectivity. An example of a tripartite plant virus is alfalfa mosaic virus (AMV). Many plant viruses also have smaller subgenomic mRNAs that are synthesized to amplify a specific gene product.
DNA Plant Viruses
Plant viruses with a double-stranded DNA genome include Cauliflower Mosaic virus (CaMV).
Plant viruses with single-stranded DNA genomes include geminiviruses, and more specifically, include African Cassava Mosaic Virus (ACMV), Tomato Golden Mosaic Virus (TGMV), and Maize Streak Virus (MSV). Geminiviruses are subdivided on the basis of whether they infect monocots or dicots and whether their insect vector is a leafhopper or a whitefly. Subgroup I geminiviruses are leafhopper-transmitted and infect monocotyledonous plants (e.g., Wheat Dwarf Virus); Subgroup II geminiviruses are leafhopper-transmitted and infect dicotyledonous plants (e.g., Beet Curly Top Virus); and Subgroup III geminiviruses are whitefly-transmitted and infect dicotyledonous plants (e.g., Tomato Golden Mosaic Virus, TGMV, and African Cassava Mosaic Virus, ACMV).
Subgroup I and II geminiviruses have a single (monopartite) genome. Subgroup III geminiviruses have a bipartite genome. For example, Subgroup III geminiviruses TGMV and ACMV consist of two circular single-stranded DNA genomes, A and B, of ca. 2.8 kB each in size. DNA A and B of a given Subgroup III virus have little sequence similarity, except for an almost identical common region of about 200 bp. While both DNA A and DNA B are required for infection, only DNA A is necessary and sufficient for replication and DNA B encodes functions required for movement of the virus through the infected plant.
In both TGMV and ACMV, DNA A contains four open reading frames (ORFs) that are expressed in a bidirectional manner and arranged similarly. The ORFs are named according to their orientation relative to the common region, i.e., complementary (C) versus viral (V) in ACMV and leftward (L) or rightward (R) in TGMV. Thus, ORFs AL1, AL2, AL3, and AR1 of TGMV are homologous to AC1, AC2, AC3, and AV1, respectively, of ACMV. Three major transcripts have been identified in ACMV DNA A and these map to the AV1 and AC1 ORFs, separately and the AC2/AC3 ORFs together. There is experimental evidence for the function of these ORFs. Thus, in ACMV AC1 encodes a replication protein that is essential and sufficient for replication; AC2 is required for transactivation of the coat protein gene, AC3 encodes a protein that is not essential for replication but enhances viral DNA accumulation; and AV1 is the coat protein gene. Except for the essential viral replication protein (encoded by AC1 and AL1 in ACMV and TGMV, respectively), geminivirus replication relies on host replication and transcription machinery. Although geminiviruses are single-stranded plant DNA viruses, they replicate via double-stranded DNA intermediate by xe2x80x98rolling circle replicationxe2x80x99.
Viruses as Expression Vectors
Constructing plant viruses to introduce and express non-viral foreign genes in plants has been demonstrated (U.S. Pat. No. 4,855,237, WO 9534668). When the virus is a DNA virus, the constructions can be made to the virus itself. Alternatively, the virus can first be cloned into a bacterial plasmid for ease in constructing the desired viral vector with the foreign DNA. If the virus is an RNA virus, the virus is generally cloned as a cDNA and inserted into a plasmid. The DNA plasmid is then used to make all of the constructions. The RNA virus is then produced by transcribing the viral sequence of the plasmid and translation of the viral genes to produce the coat protein(s) which encapsidate the viral RNA. The cDNA of RNA viral genome can be cloned behind a heterologous plant promoter. Such a chimeric gene, called an xe2x80x98ampliconxe2x80x99, can be introduced into a plant cell and used to transcribe the viral RNA that can replicate autonomously [Sablowski et al. (1995) Proc. Natl. Acad. Sci. USA vol 92, pp 6901-6905].
Geminiviruses have many advantages as potential plant expression vectors. These include 1) replication to high copy numbers, 2) small, well-characterized genomes, 3) assembly into nucleosomes, and 4) nuclear replication and transcription. The DNA A component of these viruses is capable of autonomous replication in plant cells in the absence of DNA B. Vectors in which the coat protein ORF has been replaced by a heterologous coding sequence have been developed and the heterologous coding sequence expressed from the coat protein promoter [Hayes et al., Stability and expression of bacterial genes in replicating geminivirus vectors in plants. Nucleic Acids Res. 17:2391-403 (1989); Hayes et al., Gene amplification and expression in plants by a replicating geminivirus vector. Nature (London) 334:179-82 (1988)].
Greater than full length copies of wild-type TGMV A and B genomes were transformed into petunia [Rogers et al., Tomato golden mosaic virus A component DNA replicates autonomously in transgenic plants. Cell (Cambridge, Mass.) 45:593-600 (1986)]. Replication was reported in the primary transformants and in some of the selfed progeny consistent with its mendelian inheritance, indicating that the chromosomally-integrated master copy, not the replicon, is inherited. This suggests that gametophytic and/or developing seed tissues lack the ability to support replication. The report did not demonstrate whether the virus replicated in non-germinating seed tissue. Prior art shows that geminiviruses are not seed-transmitted in nature [Goodman, R. M. (1981) Geminivirus. J. Gen. Virol. vol. 54, p 9-21]. Thus, there was no evidence that they can replicate in gametophytic tissue or developing seed.
Tomato Golden Mosaic Virus (TGMV) DNA A was modified by replacing its coat protein coding sequence with that of NPT II or GUS reporter genes or with that of 35S:NPT II gene and a greater than full length copy of the modified viruses were transformed into tobacco [Hayes et al., Stability and expression of bacterial genes in replicating geminivirus vectors in plants. Nucleic Acids Res. 17:2391-403 (1989); Hayes et al., Gene amplification and expression in plants by a replicating geminivirus vector. Nature (London) 334:179-82 (1988)]. Leaves of transgenic plants showed that the high levels of the reporter enzymes was gene copy number-dependent. However, replication of the vector and reporter gene expression were not reported in seed and the genetic stability of the vector in transgenic plants in subsequent generations was not reported. Use of the African Cassava Mosaic Virus (ACMV) in similar fashion has not been reported and it is not known that ACMV DNA or the replication protein(s) can be stably maintained in progeny plants and whether it can replicate in seed tissues.
In one report, a chimeric gene (in which the constitutive plant promoter, 35S, was fused to the TGMV sequence containing ORFs AL1, AL2, and AL3) was transformed into Nicotiana benthamiana. Different transgenic lines showed significant non-uniformity in the levels of 35S:AL1-3 gene expression as well as in their ability to complement viral replication [Hanley-Bowdoin et al., Functional expression of the leftward open reading frames of the A component of tomato golden mosaic virus in transgenic tobacco plants. Plant Cell 1:1057-67 (1989)]. In another report, chimeric genes (in which the constitutive plant promoter, 35S, was fused to the coding sequence of TGMV replication protein AL1) were transformed into tobacco. The expression of TGMV replication protein in the primary transformants supported the replication of a mutant genome A lacking the replication protein. [Hanley-Bowdoin et al., Expression of functional replication protein from tomato golden mosaic virus in transgenic tobacco plants. Proc. Natl. Acad. Sci. USA. 87:1446-50 (1990)]. However, neither publication reported on the genetic stability of the chimeric replication protein gene through subsequent generations nor its ability to support viral replication in seed tissue. In another report, chimeric genes (in which the constitutive plant promoter, 35S, was fused separately to the coding sequences of TGMV replication proteins AL1, AL2, and AL3) were transformed into tobacco [Hayes et al., Replication of tomato golden mosaic virus DNA B in transgenic plants expressing open reading frames (ORFs) of DNA A: requirement of ORF AL2 for production of single-stranded DNA. Nucleic Acids Res. 17:10213-22 (1989)]. The TGMV replication protein was expressed in progeny but the genetic stability of the chimeric replication protein gene through subsequent generations was not reported. Furthermore, it was not reported whether the transgenic plants will support replication in seed tissue.
In another disclosure, Rogers et al. (EP 221044) demonstrated the expression of foreign proteins in plant tissue using a modified xe2x80x9cAxe2x80x9d genome of the TGMV gemini virus. The foreign gene was inserted in place of the gene encoding the viral coat protein and the resulting plasmid transformed into plant tissue. Rogers et al. did not report tissue specific expression of the foreign protein and are silent as to the genetic stability of the transforming plasmid.
All of the reported viral vectors have a major disadvantage. They were either not shown to be stably maintained in transgenic plants and/or not practically useful. Thus, despite intense efforts to develop plant viral vectors and viruses, no commercially useful plant virus-based recombinant vectors have been developed that are heritable and capable of episomal replication and expression in desired tissue(s) of the transgenic host plant without the need for infection every generation. In fact, replication of plant viruses is expected to be detrimental to the growth and development of plant cells. For example, when greater than full length copy of TGMV genome A is introduced into plant cell one-tenth as many transgenic plants are obtained than when genome B is used or when control transformations are done [Rodgers et al., Tomato golden mosaic virus A component DNA replicates autonomously in transgenic plants. Cell (Cambridge, Mass.) 45:593-600 (1986)]. The authors suggest this may be due to expression of a gene in TGMV A DNA. Furthermore, crude extract of plants expressing tandem copies of both TGMV A and TGMV B genomes are unable to infect Nicotiana benthamiana plants. This is consistent with having a low virus titer. Thus, transgenic plants that do regenerate could be selected for low level expression of a toxic viral gene product and low level of viral replication or are silenced by the host. This is also consistent with the authors"" finding that relatively few cells initiate release of the virus, a conclusion based on their observation that most of the tissues remain viable and nonsymptomatic. Similarly, poor replication in transgenic plants containing 35S:replication protein in other reports suggests plants are either selected for poor expression of the replication protein (presumably because of its toxicity), or that the tissue-specific expression profiles of the replication gene is different from that of viral replication.
To date, no plant virus-based recombinant vectors are known that are heritable and capable of episomal replication and expression of foreign proteins in target tissue(s) of a transgenic host plant without the need for infection in every generation.
Although, the use viral vectors for gene silencing has been reported [[Covey, S. N. et al. (1997) Nature (London) 385:781-782; Kumagai et al. (1995) Proc. Natl. Acad Sci. (U.S.A.) 92:1679-1683; Kjemtrup, S. et al. (1998) Plant J. 14:91-100; Ratcliff, F. et al. (1997) Science (Washington, D.C.) 276:1558-1560; Ruiz, M. T. et al. (1998) Plant Cell 10:937-946; Baulcombe, D. C. and Angell, S. M. (1998) Virus amplicons for gene silencing in transgenic plants, PCT Int. Appl. WO 9836083], its use in transgenic plants under the control of conditional or regulated site-specific recombination has not been reported thus far. In the absence of such regulation, current viral gene silencing is constitutively on. This could be detrimental to a plant and restricts transgenic viral silencing to non-essential genes. Although the use silencing suppressors genes for overcoming silencing of transgenic genes has been demonstrated, their use in preventing silencing of transgenes in viral episomes has yet not been demonstrated.
Transgenic viral vectors for foreign protein production and/or gene silencing differ from infecting viral vectors in not requiring systemic movement. Use of constitutively expressed viral transgenes genes for viral resistance has been reported. However, conditional expression of such transgenes, preferably through conditional activation of replicon, say upon viral infection, is likely to provide a more effective control.
Conditional or regulated expression has been reported in plants [see De Veylder, L. et al., Plant Cell Physiol. 38:568-577 (1997); Gatz, C., Annu. Rev. Plant Physiol. Plant Mol. Biol. 48:89-108 (1997); Hansen, G. et al., Mol. Gen. Genet. 254:337-343 (1997); Jepson, I., PCT Int. Appl. (1997) WO 9706269 A1; Jepson, I, et al. PCT Int. Appl. (1997) WO 9711189 A2, and other references within this application]. However, when tested for stringently for basal non-specific expression, very few have been strictly specific [Odell J. T. et al., Plant Physiol. 91994) 106:447-458; van der Geest et al., Plant Physiol. (1995), 109(4), 1151-58; Ma et al., Aust. J Plant Physiol. (1998), 25(1), 53-59; Czako et al., Mol. Gen. Genet. (1992), 235(1), 33-40]. Such promoters are not suitable for some applications, such as the use of transgenes for expressing novel phytotoxic proteins, enzymes that lead to the biosynthesis of phytotoxic products, and/or gene silencing. Site-specific recombinations in plants (Odell et al., Plant Physiol. 106:447-458 (1994); Odell et al., PCT Int. Appl. (1991) WO 9109957; Surin et al., PCT Int. Appl.(1997) WO 9737012) and the reduction in the proficiency of Cre-mediated recombination by mutant lox P sites and their use in increasing the frequency of Cre-lox based integration have been reported [Albert et al., Plant J. 7:649-59 (1995); Araki et al., Nucleic Acids Res. 25:868-872 (1997)]. However, the use of the mutant sites to enhance the specificity Cre-mediated recombination in conjuction with chimeric Cre genes under the control of available regulated promoters has not been demonstrated. Thus, there is a need for an appropriately stringent, site-specific recombination system for a commercially-attractive, conditional site-specific recombination.
The present invention provides a binary transgenic viral expression system comprising:
(i) a chromosomally-integrated inactive replicon comprising:
a) cis-acting viral elements required for viral replication;
b) a target gene comprising at least one suitable regulatory sequence; and
c) site-specific sequences responsive to a site-specific recombinase; and
(ii) a chromosomally-integrated chimeric transactivating gene comprising a regulated plant promoter operably-linked to a site-specific recombinase coding sequence;
wherein expression of the chimeric transactivating gene in cells containing the inactive replicon results in the site-specific recombination, activation of replicon replication, and increased expression of the target gene.
The invention further provides that inactive replicon be derived from a geminivirus or a single stranded RNA virus.
Additionally the invention provides that the regulated plant promoter may be tissue-specific, constitutive or inducible and the wild-type or mutant, site-specific sequences responsive to a site-specific recombinase, the site-specific sequences may be lox sequences, responsive to the Cre recombinase protein.
The invention further provides a method of altering the levels of a protein encoded by a target gene in a plant comprising: (i) transforming a plant with the instant viral expression system of; and (ii) growing the transformed plant seed under conditions wherein the protein is expressed.
Additionally the invention provides a method of altering the levels of a protein encoded by a target gene in a plant comprising:
(i) transforming a first plant with a inactive replicon to form a first primary transformant, the inactive replicon comprising:
a) cis-acting viral elements required for viral replication;
b) a target gene comprising at least one suitable regulatory sequence; and
c) site-specific sequences responsive to a site-specific recombinase,
(ii) transforming a second plant with a chimeric transactivating gene to form a second primary transformant comprising a regulated plant promoter operably-linked to a transactivating site-specific recombinase coding sequence;
(iii) growing the first and second primary transformants wherein progeny from both seeds are obtained; and
(iv) crossing the progeny of the first and second transformants wherein the target gene is expressed.
In an alternate embodiment the invention provides a method of altering the levels of a protein encoded by a target gene in a plant comprising:
(i) transforming a plant with a inactive replicon the inactive replicon comprising:
a) cis-acting viral elements required for viral replication;
b) a target gene comprising at least one suitable regulatory sequence; and
c) site-specific sequences responsive to a site-specific recombinase;
(ii) infecting the transformant with a virus containing a chimeric transactivating gene comprising a regulated plant promoter operably-linked to a transactivating site-specific recombinase coding sequence;
wherein expression of the chimeric transactivating gene in cells containing the inactive replicon results in the site-specific recombination, activation of replicon replication, and increased expression of the target gene.
In another embodiment the invention provides a binary transgenic expression system comprising an inactive transgene and a chimeric transactivating gene, the inactive transgene comprising;
i) cis-acting transcription regulatory elements inoperably-linked to the coding sequence or functional RNA, and
ii) site-specific sequences responsive to a site specific recombinase;
the chimeric transactivating gene comprising a regulated plant promoter operably-linked to a transactivating site-specific recombinase coding sequence, wherein expression of the chimeric transactivating gene in cells containing the inactive transgene results in an operable linkage of cis-acting transcription regulatory elements to the coding sequence or functional RNA through the site-specific recombination and increased expression of the target gene.
In an alternate embodiment the invention provides a binary transgenic expression system comprising:
(i) a chromosomally integrated blocking fragment bounded by site-specific sequences responsive to a site-specific recombinase; and
(ii) a chromosomally integrated inactive silencing suppresser transgene;
wherein expression of the site specific recombinase results in the site-specific recombination that activates the silencing suppressor gene.
Additionally the invention provides a transgenic viral expression system comprising:
(i) a chromosomally-integrated geminivirus proreplicon comprising:
a) cis-acting viral elements required for viral replication;
b) a target gene comprising at least one suitable regulatory sequence; and
c) flanking sequences that enable the excision of the elements of a) and b),
wherein the proreplicon lacks a functional replication gene for episomal replication;
(ii) a chromosomally-integrated chimeric trans-acting replication gene comprising a regulated plant promoter operably-linked to a geminivirus viral replication protein coding sequence; and
(iii) a dimer of the geminivirus B genome;
wherein expression of the trans-acting replication gene in cells containing the proreplicon results in the replication of the proreplicon and the B-genome, and increased expression of the target gene.
A further object of the invention is to provide a transgenic geminivirus expression system comprising:
(i) a chromosomally-integrated inactive replicon comprising:
a) cis-acting viral elements required for viral replication;
b) a target gene comprising at least one suitable regulatory sequence; and
c) site-specific sequences responsive to a site-specific recombinase;
(ii) a chromosomally-integrated chimeric transactivating gene comprising a regulated plant promoter operably-linked to a site-specific recombinase coding sequence;
(iii) a dimer of a geminivirus B genome;
wherein expression of the chimeric transactivating gene in cells containing the inactive replicon results in the site-specific recombination, activation of replicon and B-genome replication, and increased expression of the target gene.
Yet another object of the invention is to provide a method of increasing vial resistance in a plant comprising:
(i) transforming a first plant with a inactive replicon to form a first primary transformant, the inactive replicon comprising:
a) cis-acting viral elements required for viral replication;
b) viral sequences homologous to the infecting virus capable of conferring homology-dependent resistance
c) site-specific sequences responsive to a site-specific recombinase;
(ii) transforming a second plant with a chimeric transactivating gene to form a second primary transformant comprising a regulated plant promoter operably-linked to a transactivating site-specific recombinase coding sequence;
(iii) growing the first and second primary transformants wherein progeny from both seeds are obtained; and
(iv) crossing the progeny of the first and second transformants wherein the viral sequences homologous to the infecting virus are expressed, conveying viral resistance to the plant.
The invention additionally provides a ternary expression system comprising: a) a first recombinase element comprising a first promoter operably linked to a sequence encoding a first recombinase; b) a second recombinase element comprising a second promoter, a stop fragment bounded by site specific sequences responsive to the first recombinase and a sequence encoding a second recombinase wherein the presence of the stop fragment inhibits the transcription or translation of the second recombinase, and wherein the first and second recombinases are different; and c) a DNA molecule bounded by site specific sequences responsive to the second recombinase; wherein expression of the first recombinase excises the stop fragment from the second recombinase element, operably linking the second promoter and the sequence encoding the second recombinase, and wherein expression of the second recombinase results in site specific recombination within the DNA molecule bounded by site specific sequences responsive to the second recombinase.
The present invention is useful in transgenic plants for controlled replicon replication and expression of transgenes with or without replicon replication. Both components of the system are chromosomally-integrated and independently heritable. One component is an inactive replicon that is unable to replicate episomally unless a transactivating protein is provided in trans. The second component is a chimeric trans-activating gene in which the coding sequence of a transactivating protein is placed under the control of a tissue- or development-specific and/or inducible promoter. The transactivating protein can be either a viral replication protein or a site-specific recombination protein. When it is a viral replication protein(s), the inactive replicon is of the proreplicon type that lacks a functional replication protein(s) and cannot replicate episomally unless the replication protein(s) is provided in trans. When it is a site-specific recombinase, it can mediate site-specific recombination involving cognate site-specific sequence(s) in the inactive replicon to convert it into an active one capable of autonomous or cis replication. The two systems involving a replicon can be used independently or in combination.
The site-specific recombination system can also be applied to transactivation of an inactive transgene with or without involving episomal replication.
The different components of the invention are heritable independently and may be introduced together into a transgenic plant or brought together by crossing transgenic plants carrying the separate components, such as by the method to produce TopCross(copyright) high oil corn seed [U.S. Pat. No. 5,704,160]. Also provided are methods of making the expression cassettes and methods of using them to produce transformed plant cells having an altered genotype and/or phenotype.